Human ETS family member, ELF3

ABSTRACT

Human ELF3 polypeptides and DNA (RNA) encoding such ELF3 and a procedure for producing such polypeptides by recombinant techniques is disclosed. Also disclosed are methods for utilizing such ELF3 for the diagnosis and treatment of cancers, in particular prostate, breast, lung or other epithelial tumors, among others.

This application claims benefit of U.S. Provisional application60/028,791, filed Oct. 31, 1996.

This invention relates, in part, to newly identified polynucleotides andpolypeptides; variants and derivatives of the polynucleotides andpolypeptides; processes for making the polynucleotides and thepolypeptides, and their variants and derivatives; agonists andantagonists of the polypeptides; and uses of the polynucleotides,polypeptides, variants, derivatives, agonists and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of an ETS family member, hereinafterreferred to as "ELF3".

BACKGROUND OF THE INVENTION

This invention relates to newly identified polynucleotides, polypeptidesencoded by such polynucleotides, the use of such polynucleotides andpolypeptides, as well as the production of such polynucleotides andpolypeptides. More particularly, the polypeptides of the presentinvention are of the ETS family. The invention also relates toinhibiting or activating the action of such polypeptides.

The v-ets oncogene was first described as part of the gag-myb-ets fusionprotein produced by the avian retrovirus E26. Nunn et al., Nature, 1983,306: 391-395. The cellular homolog of v-ets is Ets1 which is atranscription factor that binds to a GGA(A/T) core sequence in thepromoters and enhancers of various genes and viruses. Waslyk et al.,Eur. J Biochem. 1983, 211: 7-18; Tymms, M. J. and Kola, I, Mol. Rep.Dev., 1994, 39: 208-214. ETS proteins are believed to play a role in awide range of biological processes including hematopoiesis (Scott etal., Science, 1994, 265: 1573-1577), regulation of lymphoid cellfunction (Muthusamy et al., Nature, 1995, 377: 639-642), angiogenesis(Wernert et al., Am. J Pathol., 1992, 140: 119-127; Vandenbunder et al.,Invasion Metastasis, 1994, 14: 198-209), various developmental processessuch as organogenesis, branching morphogenesis (Kola et al., Proc. NatlAcad. Sci. USA, 1993, 90: 7588-7592) and skeletal development (Sumarsonoet al., Nature, 1996, 379: 534-537).

Many of the ETS family proteins have been shown to be nuclearoncoproteins and there is also evidence for tumor suppression activity.Suzuki et al., Proc. Natl Acad. Sci. USA, 1995, 92: 4442-4446. v-Etsinduces erthroblastosis in chickens (Metz, T. and Graf, T., Genes Dev.,1991, 5: 369-380) and cellular ETS-family proteins can be activated byproviral insertion in mice (Bellacosa et al., J. Virol., 1994, 68:2320-2330) and most significantly by chromosomal translocations inhumans. ETS1, ETS2 and ERG are proto-oncogenes that have transformingand mitogenic properties in NIH3T3 cells. Seth et al., Proc. Natl Acad.Sci USA, 1989, 86: 7833-7837; Seth, A. and Papas, T. S., Oncogene, 1990,5: 1761-1768; Hart et al., Oncogene, 1995, 10: 1423-1430. Translocationsinvolving ETS-family members which result in fusion proteins that havealtered biological properties can give rise to cancers in humans. ERGand its closely related homologue ERGB/FLI-1 have been shown to bedisrupted in t(11;22) or t(21;22) chromosomal translocations that arediagnostic of Ewings sarcomas and other primitive neuroectodermaltumors. Delattre et al., N. Engl. J Med., 1994, 331: 294-299; Sorensonet al., Nat. Genet., 1994, 6: 146-151. ERG is also involved in t(16;21)translocations associated with acute myeloid leukemia. Shimizu et al.,Proc. Natl Acad. Sci. USA, 1993, 90: 10280-10284. ETS family member TELis involved in t(5;12) translocations in chronic myelomonocytic leukemia(Golub et al., Cell, 1994, 77: 307-316) and t(12;21) translocations inacute lymphoblastic leukemia (Golub et al., Proc. Natl Acad. Sci. USA,1995, 92: 4917-4921).

Thus these proteins have an established, proven history as diagnosticagents for various types of cancer. Clearly there is a need foridentification and characterization of further ETS family members whichcan play a role in diagnosing, preventing, ameliorating or correctingdysfunctions or diseases, including, but not limited to, cancer, amongothers.

SUMMARY OF THE INVENTION

Toward these ends, and others, it is an object of the present inventionto provide polypeptides, inter alia, that have been identified as novelELF3 by homology between the amino acid sequence set out in FIG. 1 andknown amino acid sequences of other proteins such as ELF1 and ELF2 andthe DNA binding domain of Drosophila E74A.

It is a further object of the invention, moreover, to providepolynucleotides that encode ELF3, particularly polynucleotides thatencode the polypeptide of SEQ ID NO:2.

In a particularly preferred embodiment of this aspect of the invention,the polynucleotide comprises the region encoding human ELF3 in thesequence set out in FIG. 1.

In accordance with this aspect of the present invention, there isprovided an isolated nucleic acid molecule encoding a mature polypeptideexpressible from the human cDNA contained in ATCC Deposit No. 98238.

In accordance with this aspect of the invention, there are providedisolated nucleic acid molecules encoding human ELF3, including mRNAs,cDNAs, genomic DNAs and fragments and, in further embodiments of thisaspect of the invention, biologically, diagnostically, clinically ortherapeutically useful variants, analogs or derivatives thereof,including fragments of the variants, analogs and derivatives.

Among the particularly preferred embodiments of this aspect of theinvention are naturally occurring allelic variants of human ELF3.

It also is an object of the invention to provide ELF3 polypeptides,particularly human ELF3 polypeptides, that may be employed fordiagnostic purposes, for example, in the early diagnosis of cancer,among others.

In accordance with this aspect of the invention, there are providednovel polypeptides of human origin referred to herein as ELF3 as well asbiologically, diagnostically or therapeutically useful fragments,variants and derivatives thereof, variants and derivatives of thefragments, and analogs of the foregoing.

Among the particularly preferred embodiments of this aspect of theinvention are variants of human ELF3 encoded by naturally occurringalleles of the human ELF3 gene.

In accordance with another aspect of the present invention, there areprovided methods of screening for compounds which bind to and activateor inhibit activation of the polypeptides of the present invention.

It is another object of the invention to provide a process for producingthe aforementioned polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing. In a preferred embodiment of this aspect of the inventionthere are provided methods for producing the aforementioned ELF3polypeptides comprising culturing host cells having expressiblyincorporated therein an exogenously-derived human ELF3-encodingpolynucleotide under conditions for expression of human ELF3 in thehost; expressing the ELF3 polypeptide; and then recovering the expressedpolypeptide.

In accordance with another object the invention, there are providedproducts, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides for research,biological, clinical and therapeutic purposes, inter alia.

In accordance with certain preferred embodiments of this aspect of theinvention, there are provided products, compositions and methods, interalia, for, among other things: assessing ELF3 expression in cells bydetermining ELF3 polypeptides or ELF3-encoding MRNA; to diagnose canceramong others, in vitro, ex vivo or in vivo by exposing cells to ELF3polypeptides or polynucleotides as disclosed herein; assaying geneticvariation and aberrations, such as defects, in ELF3 genes; andadministering an ELF3 polypeptide or polynucleotide to an organism toaugment ELF3 function or remediate ELF3 dysfunction.

In accordance with still another embodiment of the present invention,there is provided a process of using such activating compounds tostimulate the polypeptide of the present invention for the treatment ofconditions related to the under-expression of ELF3.

In accordance with another aspect of the present invention, there isprovided a process of using such inhibiting compounds for treatingconditions associated with over-expression of the ELF3.

In accordance with yet another aspect of the present invention, there isprovided non-naturally occurring synthetic, isolated and/or recombinantELF3 polypeptides which are fragments, consensus fragments and/orsequences having conservative amino acid substitutions of at least onedomain of the ELF3 polypeptide of the present invention, such that thepolypeptide may bind ELF3 ligands, or which may also modulate,quantitatively or qualitatively, ELF3 ligand binding.

In accordance with still another aspect of the present invention, thereare provided synthetic or recombinant ELF3 polypeptides, conservativesubstitution and derivatives thereof, antibodies thereto, anti-idiotypeantibodies, compositions and methods that can be useful as potentialmodulators of ELF3 function, by binding to ligands or modulating ligandbinding, due to their expected biological properties, which may be usedin diagnostic, therapeutic and/or research applications.

It is still another object of the present invention to providesynthetic, isolated or recombinant polypeptides which are designed toinhibit or mimic various ELF3 or fragments thereof.

In accordance with certain preferred embodiments of this and otheraspects of the invention, there are provided probes that hybridize tohuman ELF3 sequences.

In certain additional preferred embodiments of this aspect of theinvention, there are provided antibodies against ELF3 polypeptides. Incertain particularly preferred embodiments in this regard, theantibodies are highly selective for human ELF3.

In accordance with another aspect of the present invention, there areprovided ELF3 agonists. Among preferred agonists are molecules thatmimic ELF3, that bind to ELF3-binding molecules or receptor molecules,and that elicit or augment ELF3-induced responses. Also among preferredagonists are molecules that interact with ELF3 or ELF3 polypeptides, orwith other modulators of ELF3 activities, and thereby potentiate oraugment an effect of ELF3 or more than one effect of ELF3.

In accordance with yet another aspect of the present invention, thereare provided ELF3 antagonists. Among preferred antagonists are thosewhich mimic ELF3 so as to bind to an ELF3 receptor or binding molecules,but not elicit an ELF3-induced response or more than one ELF3-inducedresponse. Also among preferred antagonists are molecules that bind to orinteract with ELF3 so as to inhibit an effect of ELF3 or more than oneeffect of ELF3 or which prevent expression of ELF3.

In a further aspect of the invention, there are provided compositionscomprising an ELF3 polynucleotide or an ELF3 polypeptide foradministration to cells in vitro, to cells ex vivo and to cells in vivo,or to a multicellular organism. In certain particularly preferredembodiments of this aspect of the invention, the compositions comprisean ELF3 polynucleotide for expression of an ELF3 polypeptide in a hostorganism for treatment of disease. Particularly preferred in this regardis expression in a human patient for treatment of a dysfunctionassociated with aberrant endogenous activity of ELF3.

Other objects, features, advantages and aspects of the present inventionwill become apparent to those of skill in the art from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings depict certain embodiments of the invention. Theyare illustrative only and do not limit the invention otherwise disclosedherein.

FIGS. 1A and 1B shows the nucleotide and deduced amino acid sequence ofhuman ELF3.

FIG. 1A shows the nucleotide sequence of human ELF3 (SEQ ID NO:1). Startand stop codons of the nucleic acid sequence are indicated in bold.

FIG. 1B1 and B2 shows the deduced amino acid sequence of human ELF3 (SEQID NO:2). The ETS binding domain of the amino acid sequence is indicatedin bold. Amino acids of the PEST region are indicated by underlining.

GLOSSARY

The following illustrative explanations are provided to facilitateunderstanding of certain terms used frequently herein, particularly inthe examples. The explanations are provided as a convenience and are notmeant to limit the invention.

"Digestion" of DNA refers to catalytic cleavage of a DNA with an enzymesuch as, but not limited to, a restriction enzyme that acts only atcertain sequences in the DNA. The various restriction enzymes referredto herein are commercially available and their reaction conditions,cofactors and other requirements for use are known and routine to theskilled artisan.

For analytical purposes, typically, 1 microgram of plasmid or DNAfragment is digested with about 2 units of enzyme in about 20microliters of reaction buffer. For the purpose of isolating DNAfragments for plasmid construction, typically 5 to 50 micrograms of DNAare digested with 20 to 250 units of enzyme in proportionately largervolumes.

Appropriate buffers and substrate amounts for particular restrictionenzymes are described in standard laboratory manuals, such as thosereferenced below, and they are specified by commercial suppliers.

Incubation times of about 1 hour at 37° C. are ordinarily used, butconditions may vary in accordance with standard procedures, thesupplier's instructions and the particulars of the reaction. Afterdigestion, reactions may be analyzed, and fragments may be purified byelectrophoresis through an agarose or polyacrylamide gel, using wellknown methods that are routine for those skilled in the art.

"Genetic element" generally means a polynucleotide comprising a regionthat encodes a polypeptide or a region that regulates replication,transcription or translation or other processes important to expressionof the polypeptide in a host cell, or a polynucleotide comprising both aregion that encodes a polypeptide and a region operably linked theretothat regulates expression.

Genetic elements may be comprised within a vector that replicates as anepisomal element; that is, as a molecule physically independent of thehost cell genome. They may be comprised within mini-chromosomes, such asthose that arise during amplification of transfected DNA by methotrexateselection in eukaryotic cells. Genetic elements also may be comprisedwithin a host cell genome, not in their natural state but, rather,following manipulation such as isolation, cloning and introduction intoa host cell in the form of purified DNA or in a vector, among others.

"Isolated" means altered "by the hand of man" from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both.

For example, a naturally occurring polynucleotide or a polypeptidenaturally present in a living animal in its natural state is not"isolated," but the same polynucleotide or polypeptide separated fromthe coexisting materials of its natural state is "isolated", as the termis employed herein. For example, with respect to polynucleotides, theterm isolated means that it is separated from the chromosome and cell inwhich it naturally occurs.

As part of or following isolation, such polynucleotides can be joined toother polynucleotides, such as DNAs, for mutagenesis, to form fusionproteins, and for propagation or expression in a host, for instance. Theisolated polynucleotides, alone or joined to other polynucleotides suchas vectors, can be introduced into host cells, in culture or in wholeorganisms. Introduced into host cells in culture or in whole organisms,such DNAs still would be isolated, as the term is used herein, becausethey would not be in their naturally occurring form or environment.Similarly, the polynucleotides and polypeptides may occur in acomposition, such as a media, formulations, solutions for introductionof polynucleotides or polypeptides, for example, into cells,compositions or solutions for chemical or enzymatic reactions, forinstance, which are not naturally occurring compositions, and, thereinremain isolated polynucleotides or polypeptides within the meaning ofthat term as it is employed herein.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo or more polynucleotides, which most often are double stranded DNAs.Techniques for ligation are well known to the art and protocols forligation are described in standard laboratory manuals and references,such as, for instance, Sambrook et al., MOLECULAR CLONING, A LABORATORYMANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989, hereinafter referred to as Sambrook et al.

"Oligonucleotide(s)" refers to relatively short polynucleotides. Oftenthe term refers to single-stranded deoxyribonucleotides, but it canrefer as well to single-or double-stranded ribonucleotides, RNA:DNAhybrids and double-stranded DNAs, among others.

Oligonucleotides, such as single-stranded DNA probe oligonucleotides,often are synthesized by chemical methods, such as those implemented onautomated oligonucleotide synthesizers. However, oligonucleotides can bemade by a variety of other methods, including in vitro recombinantDNA-mediated techniques and by expression of DNAs in cells andorganisms.

Initially, chemically synthesized DNAs typically are obtained without a5' phosphate. The 5' ends of such oligonucleotides are not substratesfor phosphodiester bond formation by ligation reactions that employ DNAligases typically used to form recombinant DNA molecules. Where ligationof such oligonucleotides is desired, a phosphate can be added bystandard techniques, such as those that employ a kinase and ATP.

The 3' end of a chemically synthesized oligonucleotide generally has afree hydroxyl group and, in the presence of a ligase, such as T4 DNAligase, will readily form a phosphodiester bond with a 5' phosphate ofanother polynucleotide, such as another oligonucleotide. As is wellknown, this reaction can be prevented selectively, where desired, byremoving the 5' phosphates of the other polynucleotide(s) prior toligation.

"Plasmids" are genetic elements that are stably inherited without beinga part of the chromosome of their host cell. They may be comprised ofDNA or RNA and may be linear or circular. Plasmids code for moleculesthat ensure their replication and stable inheritance during cellreplication and may encode products of considerable medical,agricultural and environmental importance. For example, they code fortoxins that greatly increase the virulence of pathogenic bacteria. Theycan also encode genes that confer resistance to antibiotics. Plasmidsare widely used in molecular biology as vectors used to clone andexpress recombinant genes. Plasmids generally are designated herein by alower case "p" preceded and/or followed by capital letters and/ornumbers, in accordance with standard naming conventions that arefamiliar to those of skill in the art. Starting plasmids disclosedherein are either commercially available, publicly available, or can beconstructed from available plasmids by routine application of wellknown, published procedures. Many plasmids and other cloning andexpression vectors that can be used in accordance with the presentinvention are well known and readily available to those of skill in theart. Moreover, those of skill readily may construct any number of otherplasmids suitable for use in the invention. The properties, constructionand use of such plasmids, as well as other vectors, in the presentinvention will be readily apparent to those of skill from the presentdisclosure.

"Polynucleotide(s)" generally refers to any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. Thus, for instance, polynucleotides as used herein refersto, among others, single- and double-stranded DNA, DNA that is a mixtureof single- and double-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded or a mixture of single- and double-strandedregions.

In addition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide.

As used herein, the term polynucleotide includes DNAs or RNAs asdescribed above that contain one or more modified bases. Thus, DNAs orRNAs with backbones modified for stability or for other reasons arepolynucleotides as that term is intended herein. Moreover, DNAs or RNAscomprising unusual bases, such as inosine, or modified bases, such astritylated bases, to name just two examples, are polynucleotides as theterm is used herein.

It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide, as it is employed herein,embraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia simple andcomplex cells.

"Polypeptides", as used herein, includes all polypeptides as describedbelow. The basic structure of polypeptides is well known and has beendescribed in innumerable textbooks and other publications in the art. Inthis context, the term is used herein to refer to any peptide or proteincomprising two or more amino acids joined to each other in a linearchain by peptide bonds. As used herein, the term refers to both shortchains, which also commonly are referred to in the art as peptides,oligopeptides and oligomers, for example, and to longer chains, whichgenerally are referred to in the art as proteins, of which there aremany types.

It will be appreciated that polypeptides often contain amino acids otherthan the 20 amino acids commonly referred to as the 20 naturallyoccurring amino acids, and that many amino acids, including the terminalamino acids, may be modified in a given polypeptide, either by naturalprocesses, such as processing and other post-translationalmodifications, or by chemical modification techniques which are wellknown to the art. Even the common modifications that occur naturally inpolypeptides are too numerous to list exhaustively here, but they arewell described in basic texts and in more detailed monographs, as wellas in a voluminous research literature, and thus are well known to thoseof skill in the art.

Examples of known modifications which may be present in polypeptides ofthe present invention include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cystine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination. Such modifications are well known to those of skill andhave been described in great detail in the scientific literature.Several particularly common modifications such as glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation are described in basic texts such asPROTEINS--STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York, 1993. Detailed reviews are alsoavailable on this subject. See e.g. Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pages 1-12 inPOSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, Ed.,Academic Press, New York, 1983; Seifter et al., Meth. Enzymol., 1990,182: 626-646 and Rattan et al., Ann. N. Y. Acad. Sci., 1992, 663: 48-62.

It will be appreciated, as is well known and as noted above, thatpolypeptides are not always entirely linear. For instance, polypeptidesmay be branched as a result of ubiquitination, and they may be circular,with or without branching, generally as a result of posttranslationevents, including natural processing event and events brought about byhuman manipulation which do not occur naturally. Circular, branched andbranched circular polypeptides may be synthesized by non-translationnatural process and by entirely synthetic methods, as well.

Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.In fact, blockage of the amino or carboxyl group in a polypeptide, orboth, by a covalent modification, is common in naturally occurring andsynthetic polypeptides and such modifications may be present inpolypeptides of the present invention, as well. For instance, the aminoterminal residue of polypeptides made in E. coli, prior to processing,almost invariably will be N-formylmethionine.

The modifications that occur in a polypeptide often will be a functionof how it is made. For polypeptides made by expressing a cloned gene ina host, for instance, the nature and extent of the modifications inlarge part will be determined by the host cell's posttranslationalmodification capacity and the modification signals present in thepolypeptide amino acid sequence. For instance, as is well known,glycosylation often does not occur in bacterial hosts such as E. coli.Accordingly, when glycosylation is desired, a polypeptide should beexpressed in a glycosylating host, generally a eukaryotic cell. Insectcells often carry out the same posttranslational glycosylations asmammalian cells and, for this reason, insect cell expression systemshave been developed to express efficiently mammalian proteins having thenative patterns of glycosylation, inter alia. Similar considerationsapply to other modifications.

It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given polypeptide.Also, a given polypeptide may contain many types of modifications.

In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

"Variant(s)" of polynucleotides or polypeptides, as the term is usedherein, are polynucleotides or polypeptides that differ from a referencepolynucleotide or polypeptide, respectively. Variants in this sense aredescribed below and elsewhere in the present disclosure in greaterdetail.

Variants include polynucleotides that differ in nucleotide sequence fromanother, reference polynucleotide. Generally, differences are limited sothat the nucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical.

As noted below, changes in the nucleotide sequence of the variant may besilent. That is, they may not alter the amino acids encoded by thepolynucleotide. Where alterations are limited to silent changes of thistype, a variant will encode a polypeptide with the same amino acidsequence as the reference. As also noted below, changes in thenucleotide sequence of the variant may alter the amino acid sequence ofa polypeptide encoded by the reference polynucleotide. Such nucleotidechanges may result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptide encoded by the referencesequence, as discussed below.

Variants also include polypeptides that differ in amino acid sequencefrom another, reference polypeptide. Generally, differences are limitedso that the sequences of the reference and the variant are closelysimilar overall and, in many regions, identical.

A variant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, deletions, fusions andtruncations, which may be present in any combination.

"Fusion protein" as the term is used herein, is a protein encoded bytwo, often unrelated, fused genes or fragments thereof. EP-A-O 464 533(Canadian counterpart 2045869) discloses fusion proteins comprisingvarious portions of constant region of immunoglobin molecules togetherwith another human protein or part thereof. In many cases, employing animmunoglobulin Fc regions as a part of a fusion protein is advantageousfor use in therapy and diagnosis resulting in, for example, improvedpharmacokinetic properties (EP-A 0232 262). On the other hand, for someuses it would be desirable to be able to delete the Fc part after thefusion protein has been expressed, detected and purified. Accordingly,it may be desirable to link the components of the fusion protein with achemically or enzymatically cleavable linking region. This is the casewhen the Fc portion proves to be a hindrance to use in therapy anddiagnosis, for example, when the fusion protein is to be used as anantigen for immunizations. In drug discovery, for example, humanproteins, such as, shIL5-α have been fused with Fc portions for use inhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al, Journal of Molecular Recognition, 1995, 8: 52-58; andK. Johanson et al., The Journal of Biological Chemistry, 1995,270(16):9459-9471.

Thus, this invention also relates to genetically engineered solublefusion proteins comprised of ELF3, or a portion thereof, and of variousportions of the constant regions of heavy or light chains ofimmunoglobulins of various subclasses (IgG, IgM, IgA, IgE). Preferred asan immunoglobulin is the constant part of the heavy chain of human IgG,particularly IgG1, where fusion takes place at the hinge region. In aparticular embodiment, the Fc part can be removed simple byincorporation of a cleavage sequence which can be cleaved with bloodclotting factor Xa. Furthermore, this invention relates to processes forthe preparation of these fusion proteins by genetic engineering, and tothe use thereof for diagnosis and therapy. A further aspect of theinvention also relates to polynucleotides encoding such fusion proteins.

Transcription factors are particularly useful in the preparation offusion proteins. Such factors are generally characterized as possessingat least two distinct structural regions: DNA binding domain(s) andtransactivation domains(s). The invention contemplates the use of one ormore of these regions as components of a fusion protein. Examples ofsuch fusion protein technology can be found in WO94/29458 andWO94/22914.

"Binding molecules" refer to molecules that specifically bind to orinteract with polypeptides of the present invention. Such bindingmolecules are a part of the present invention. Binding molecules mayalso be non-naturally occurring, such as antibodies and antibody-derivedreagents that bind specifically to polypeptides of the invention.

As known in the art, "similarity" between two polypeptides is determinedby comparing the amino acid sequence and its conserved amino acidsubstitutes of one polypeptide to the sequence of a second polypeptide.Moreover, also known in the art is "identity" which means the degree ofsequence relatedness between two polypeptide or two polynucleotidesequences as determined by the identity of the match between two stringsof such sequences. Both identity and similarity can be readilycalculated (COMPUTATIONAL MOLECULAR BIOLOGY, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; BIOCOMPUTING: INFORMATICS AND GENOMEPROJECTS, Smith, D. W., ed., Academic Press, New York, 1993; COMPUTERANALYSIS OF SEQUENCE DATA, PART I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; SEQUENCE ANALYSIS IN MOLECULARBIOLOGY, von Heinje, G., Academic Press, 1987; and SEQUENCE ANALYSISPRIMER, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991). A number of methods exist to measure identity and similaritybetween two polynucleotide or polypeptide sequences and the terms"identity" and "similarity" are well known to skilled artisans (Carillo,H., and Lipton, D., SIAM J Applied Math., 1988, 48: 1073). Methodscommonly employed to determine identity or similarity between twosequences include, but are not limited to, those disclosed in GUIDE TOHUGE COMPUTERS, Martin J. Bishop, ed., Academic Press, San Diego, 1994,and Carillo, H., and Lipton, D., SIAM J. Applied Math., 1988, 48: 1073.Preferred methods to determine identity are designed to give the largestmatch between the two sequences tested. Methods to determine identityand similarity are also codified in computer programs. Preferredcomputer program methods to determine identity and similarity betweentwo sequences include, but are not limited to, GCG program package(Devereux, J., et al., Nucleic Acids Research, 1984, 12(1):387), BLASTP,BLASTN, and FASTA (Atschul, S. F. et al., J. Molec. Biol., 1990, 215:403).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel ELF3 polypeptides andpolynucleotides, among other things, as described in greater detailbelow. In particular, the invention relates to polypeptides andpolynucleotides of a novel human ELF3, which is related by amino acidsequence homology to ELF1 and ELF2 and the DNA binding domain ofDrosophila E74A. The invention relates especially to ELF3 having thenucleotide and amino acid sequences set out in FIG. 1 and to thenucleotide sequences of the human cDNA in ATCC Deposit No. 98238, hereinreferred to as "the deposited clone" or as the "cDNA of the depositedclone", and amino acid sequences encoded thereby. It will be appreciatedthat the nucleotide and amino acid sequences set out in FIG. 1 areobtained by sequencing the cDNA of the deposited clone. Hence, thesequence of the deposited clone is controlling as to any discrepanciesbetween the two and any reference to the sequences of FIG. 1 includes areference to the sequence of the human cDNA of the deposited clone.

Polynucleotides

In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the ELF3 polypeptidehaving the deduced amino acid sequence of FIG. 1.

Using the information provided herein, such as the polynucleotidesequence set out in FIG. 1, a polynucleotide of the present inventionencoding human ELF3 may be obtained using standard cloning and screeningprocedures, such as those for cloning cDNAs using MRNA from cells fromprostate tissue as starting material. Illustrative of the invention, thepolynucleotide set out in FIG. 1 was discovered in a lung cDNA libraryusing a probe from a prostate cDNA library identified by expressedsequence tag (EST) analysis (Adams, M. D., et al. Science, (1991), 252:1651-1656; Adams, M. D. et al., Nature, (1992), 355: 632-634; Adams, M.D., et al., Nature, (1995), 377 Supp:3-174). More specifically, a knownETS DNA binding domain sequence derived from a prostate cDNA library wasused to screen the lung cDNA library. This sequence is highly conservedamongst members of the ETS family and serves as a diagnostic forclassification as a member of this family.

Human ELF3 of the invention is structurally related to other proteins ofthe ETS family, as shown by the results of sequencing the cDNA encodinghuman ELF3 in the deposited clone. The DNA binding domain of ELF3 placesit in the E74 ETS DNA binding domain subfamily. ELF 3 show homology withother human ETS family proteins in the characteristic ETS DNA bindingdomain and shows the highest homology with ELF 1 and ELF 2 which havebeen assigned these gene symbols on the basis of the high identity tothe DNA binding domain of Drosophila E74A. ELF3 has 48% identity withELFI, 49% identity with ELF2 and 50% identity with E74. The cDNAsequence obtained is set out in FIG. 1 and also SEQ ID NO: 1. Itcontains an open reading frame encoding a protein of 371 amino acidswith a deduced molecular weight of 41,454 Da. Estimated size of theprotein by SDS-PAGE is 42 kDa. Amino acids 42 to 135 of SEQ ID NO:2represent the N-terminal domain and show homology with ETS1 and ETS2.Amino acids 209 to 221 of SEQ ID NO:2 represent the consensus PESTmotif. Amino acids 274 to 353 of SEQ ID NO:2 represent the ETSDNA-binding domain.

Polynucleotides of the present invention may be in the form of RNA, suchas mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the antisense strand.

The coding sequence which encodes the polypeptide may be identical tothe coding sequence of the polynucleotide shown in FIG. 1, SEQ ID NO: 1.It may also be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, also encodesthe polypeptide of FIG. 1, SEQ ID NO: 2.

Polynucleotides of the present invention which encode the polypeptide ofFIG. 1 may include, but are not limited to, the coding sequence for themature polypeptide, by itself; the coding sequence for the maturepolypeptide and additional coding sequences; and the coding sequence ofthe mature polypeptide, with or without the aforementioned additionalcoding sequences, together with additional, non-coding sequences.Examples of additional coding sequence include, but are not limited to,sequences such as those encoding a leader or secretory sequence, such asa preprotein, proprotein or preproprotein sequence. Examples ofadditional non-coding sequences include, but not limited to, introns andnon-coding 5' and 3' sequences, such as the transcribed, non-translatedsequences that play a role in transcription and MRNA processing,including splicing and polyadenylation signals, for example, forribosome binding and stability of mRNA. Coding sequences which provideadditional functionalities may also be incorporated into thepolypeptide. Thus, for instance, the polypeptide may be fused to amarker sequence, such as a peptide, which facilitates purification ofthe fused polypeptide. In certain preferred embodiments of this aspectof the invention, the marker sequence is a hexa-histidine peptide, suchas that provided in the pQE vector (Qiagen, Inc.). As described in Gentzet al., Proc. Natl. Acad. Sci., USA, 1989, 86: 821-824, for instance,hexa-histidine provides for convenient purification of the fusionprotein. The HA tag corresponds to an epitope derived of influenzahemagglutinin protein, which has been described by Wilson et al., Cell,1984, 37: 767. Many other such tags are commercially available.

In accordance with the foregoing, the term "polynucleotide encoding apolypeptide" as used herein encompasses polynucleotides which include,by virtue of the redundancy of the genetic code, any sequence encoding apolypeptide of the present invention, particularly the human ELF3 havingthe amino acid sequence set out in FIG. 1. The term also encompassespolynucleotides that include a single continuous region or discontinuousregions encoding the polypeptide (for example, interrupted by introns)together with additional regions, that also may contain coding and/ornon-coding sequences.

The present invention further relates to variants of the herein abovedescribed polynucleotides which encode for fragments, analogs andderivatives of the polypeptide having the deduced amino acid sequence ofFIG. 1. A variant of the polynucleotide may be a naturally occurringvariant such as a naturally occurring allelic variant, or it may be avariant that is not known to occur naturally. Such non-naturallyoccurring variants of the polynucleotide may be made by mutagenesistechniques, including those applied to polynucleotides, cells ororganisms.

Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions, deletions or additions may involve one ormore nucleotides. The variants may be altered in coding or non-codingregions or both. Alterations in the coding regions may produceconservative or non-conservative amino acid substitutions, deletions oradditions.

Among the particularly preferred embodiments of the invention in thisregard are polynucleotides encoding polypeptides having the amino acidsequence of ELF3 set out in FIG. 1; variants, analogs, derivatives andfragments thereof, and fragments of the variants, analogs andderivatives.

Further particularly preferred in this regard are polynucleotidesencoding ELF3 variants, analogs, derivatives and fragments, andvariants, analogs and derivatives of the fragments, which have the aminoacid sequence of the ELF3 polypeptide of FIG. 1 in which several, a few,5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted,deleted or added, in any combination. Especially preferred among theseare silent substitutions, additions and deletions, which do not alterthe properties and activities of the ELF3. Also especially preferred inthis regard are conservative substitutions. Most highly preferred arepolynucleotides encoding polypeptides having the amino acid sequence ofFIG. 1, without substitutions.

Further preferred embodiments of the invention are polynucleotides thatare greater than 50% identical to a polynucleotide encoding the ELF3polypeptide having the amino acid sequence set out in FIG. 1, andpolynucleotides which are complementary to such polynucleotides. Morehighly preferred are polynucleotides that comprise a region that is atleast 60-80% identical to a polynucleotide encoding the ELF3 polypeptideof the human cDNA of the deposited clone and polynucleotidescomplementary thereto. In this regard, polynucleotides at least 90%identical to the same are particularly preferred, and those with atleast 95% are more particularly preferred. Furthermore, those with atleast 97% are highly preferred and those with at least 98-99% are morehighly preferred, with at least 99% being the most preferred.

Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame biological function or activity as the mature polypeptide encodedby the cDNA of FIG. 1.

The present invention further relates to polynucleotides that hybridizeto the herein above-described sequences. In this regard, the presentinvention especially relates to polynucleotides which hybridize understringent conditions to the herein above-described polynucleotides. Asherein used, the term "stringent conditions" means hybridization willoccur only if there is at least 95% and preferably at least 97% identitybetween the sequences.

As discussed additionally herein regarding polynucleotide assays of theinvention, for instance, polynucleotides of the invention as discussedabove, may be used as hybridization probes for cDNA and genomic DNA, toisolate full-length cDNAs and genomic clones encoding ELF3 and toisolate cDNA and genomic clones of other genes that have a high sequencesimilarity to the human ELF3 gene. Such probes generally will compriseat least 15 nucleotides. Preferably, such probes will have at least 30nucleotides and may have at least 50 nucleotides. Particularly preferredprobes will range between 30 and 50 nucleotides.

For example, the coding region of the ELIF3 gene may be isolated byscreening using the known DNA sequence to synthesize an oligonucleotideprobe. A labeled oligonucleotide having a sequence complementary to thatof a gene of the present invention is then used to screen a library ofhuman cDNA, genomic DNA or mRNA to determine the members of the libraryto which the probe hybridizes to.

The polynucleotides and polypeptides of the present invention may beemployed as research reagents and materials for discovery of treatmentsand diagnostics to human disease, as further discussed herein relatingto polynucleotide assays.

The polynucleotides may encode a polypeptide which is the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature polypeptide (when the mature form has more thanone polypeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, may facilitateprotein trafficking, may prolong or shorten protein half-life or mayfacilitate manipulation of a protein for assay or production, amongother things. As generally is the case in situ, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

In sum, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

Deposited materials

A deposit containing a human ELF3 cDNA has been deposited with theAmerican Type Culture Collection, 12301 Park Lawn Drive, Rockville, Md.20852, USA, on Nov. 1, 1996, and assigned ATCC Deposit No. 98238. Thehuman cDNA deposit is referred to herein as "the deposited clone" or as"the cDNA of the deposited clone."

The deposited material is an E. coli DH10B pGEMT ELF3 that contains thefull length ELF3 cDNA.

The deposit has been made under the terms of the Budapest Treaty on theinternational recognition of the deposit of micro-organisms for purposesof patent procedure. The strain will be irrevocably and withoutrestriction or condition released to the public upon the issuance of apatent. The deposit is provided merely as convenience to those of skillin the art and is not an admission that a deposit is required forenablement, such as that required under 35 U.S.C. §112.

The sequence of the polynucleotides contained in the deposited material,as well as the amino acid sequence of the polypeptide encoded thereby,are controlling in the event of any conflict with any description ofsequences herein.

A license may be required to make, use or sell the deposited materials,and no such license is hereby granted.

Polypeptides

The present invention further relates to human ELF3 polypeptide whichhas the deduced amino acid sequence of FIG. 1, SEQ ID NO: 2.

The invention also relates to fragments, analogs and derivatives ofthese polypeptides. The terms "fragment," "derivative" and "analog" whenreferring to the polypeptide of FIG. 1, mean a polypeptide which retainsessentially the same biological function or activity as suchpolypeptide, i.e. functions as an ELF3, or retains the ability to bindELF3 binding molecules even though the polypeptide does not function asan ELF3. Thus, an analog includes, for example, a proprotein which canbe activated by cleavage of the proprotein portion to produce an activemature polypeptide.

The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments, it is a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of FIG. 1 may be(i) one in which one or more of the amino acid residues are substitutedwith a conserved or non-conserved amino acid residue (preferably aconserved amino acid residue) and such substituted amino acid residuemay or may not be one encoded by the genetic code; (ii) one in which oneor more of the amino acid residues includes a substituent group; (iii)one in which the mature polypeptide is fused with another compound, suchas a compound to increase the half-life of the polypeptide (for example,polyethylene glycol); or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence or a sequence which is employed for purification of the maturepolypeptide or a proprotein sequence. Such fragments, derivatives andanalogs are deemed to be within the scope of those skilled in the artfrom the teachings herein.

Among the particularly preferred embodiments of the invention in thisregard are polypeptides having the amino acid sequence of ELF3 set outin FIG. 1, variants, analogs, derivatives and fragments thereof, andvariants, analogs and derivatives of the fragments. Further particularlypreferred embodiments of the invention in this regard are polypeptideshaving the amino acid sequence of ELF3, variants, analogs, derivativesand fragments thereof, and variants, analogs and derivatives of thefragments which retain the activity/function of ELF3.

Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and Ile; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe and Tyr.

Further particularly preferred in this regard are variants, analogs,derivatives and fragments, and variants, analogs and derivatives of thefragments, having the amino acid sequence of the ELF3 polypeptide ofFIG. 1, in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or noamino acid residues are substituted, deleted or added, in anycombination. Especially preferred among these are silent substitutions,additions and deletions, which do not alter the properties andactivities of the ELF3. Also especially preferred in this regard areconservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequence of FIG. 1 without substitutions.

The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

The polypeptides of the present invention include the polypeptide of SEQID NO: 2 (in particular the mature polypeptide) as well as polypeptideswhich have greater than 50% identity to the polypeptide of SEQ ID NO: 2and more preferably more than 50-80% similarity (more preferably greaterthan 50-80% identity) to the polypeptide of SEQ ID NO: 2 and still morepreferably at least 90% similarity (still more preferably at least 90%identity) to the polypeptide of SEQ ID NO: 2 and also include portionsof such polypeptides with such portion of the polypeptide generallycontaining at least 30 amino acids and more preferably at least 50 aminoacids.

Fragments or portions of the polypeptides of the present invention maybe employed for producing the corresponding full-length polypeptide bypeptide synthesis; therefore, the fragments may be employed asintermediates for producing the full-length polypeptides. Fragments orportions of the polynucleotides of the present invention may be used tosynthesize full-length polynucleotides of the present invention.Fragments may be "free-standing," i.e., not part of or fused to otheramino acids or polypeptides, or they may be comprised within a largerpolypeptide of which they form a part or region. When comprised within alarger polypeptide, the presently discussed fragments most preferablyform a single continuous region. However, several fragments may becomprised within a single larger polypeptide. For instance, certainpreferred embodiments relate to a fragment of an ELF3 polypeptide of thepresent comprised within a precursor polypeptide designed for expressionin a host and having heterologous pre- and pro-polypeptide regions fusedto the amino terminus of the ELF3 fragment and an additional regionfused to the carboxyl terminus of the fragment. Therefore, fragments inone aspect of the meaning intended herein, refers to the portion orportions of a fusion polypeptide or fusion protein derived from ELF3.

As representative examples of polypeptide fragments of the invention,there may be mentioned those which have from about 5-15, 10-20, 15-40,30-55, 41-75, 41-80, 41-90, 50-100, 75-100, 90-115, 100-125, and 110-113amino acids in length.

In this context "about" includes the particularly recited range andranges larger or smaller by several, a few, 5, 4, 3, 2 or 1 amino acidresidues at either extreme or at both extremes. For instance, about40-90 amino acids in this context means a polypeptide fragment of 40plus or minus several, a few, 5, 4, 3, 2 or 1 amino acid residues to 90plus or minus several a few, 5, 4, 3, 2 or 1 amino acid residues, i.e.,ranges as broad as 40 minus several amino acids to 90 plus several aminoacids to as narrow as 40 plus several amino acids to 90 minus severalamino acids. Highly preferred in this regard are the recited ranges plusor minus as many as 5 amino acids at either or at both extremes.Particularly highly preferred are the recited ranges plus or minus asmany as 3 amino acids at either or at both the recited extremes.Especially particularly highly preferred are ranges plus or minus 1amino acid at either or at both extremes or the recited ranges with noadditions or deletions. Most highly preferred of all in this regard arefragments from about 5-15, 10-20, 15-40, 30-55, 41-75, 41-80, 41-90,50-100, 75-100, 90-115, 100-125, and 110-113 amino acids long.

Among especially preferred fragments of the invention are truncationmutants of ELF3. Truncation mutants include ELF3 polypeptides having theamino acid sequence of FIG. 1, or of variants or derivatives thereof,except for deletion of a continuous series of residues (that is, acontinuous region, part or portion) that includes the amino terminus, ora continuous series of residues that includes the carboxyl terminus or,as in double truncation mutants, deletion of two continuous series ofresidues, one including the amino terminus and one including thecarboxyl terminus.

Preferred in this aspect of the invention are fragments characterized bystructural or functional attributes of ELF3. Preferred embodiments ofthe invention in this regard include fragments that comprise alpha-helixand alpha-helix forming regions ("alpha-regions"), beta-sheet andbeta-sheet-forming regions ("beta-regions"), turn and turn-formingregions ("turn-regions"), coil and coil-forming regions("coil-regions"), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of ELF3.

Among highly preferred fragments in this regard are those that compriseregions of ELF3 that contain structural regions, i.e., the DNA bindingdomain or the transactivation domain of this transcription factor. Inthis regard, the regions defined by the residues about 10 to about 20,about 40 to about 50, about 70 to about 90 and about 100 to about 113 ofFIG. 1, which all are characterized by amino acid compositions highlycharacteristic of turn-regions, hydrophilic regions, flexible-regions,surface-forming regions, and high antigenic index-regions, areespecially highly preferred regions. Such regions may be comprisedwithin a larger polypeptide or may be by themselves a preferred fragmentof the present invention, as discussed above. It will be appreciatedthat the term "about" as used in this paragraph has the meaning set outabove regarding fragments in general.

Further preferred regions are those that mediate activities of ELF3.Most highly preferred in this regard are fragments that have a chemical,biological or other activity of ELF3, including those with a similaractivity or an improved activity, or with a decreased undesirableactivity. Highly preferred in this regard are fragments that containregions that are homologs in sequence, or in position, or in bothsequence and to active regions of related polypeptides of the ETSfamily.

It will be appreciated that the invention also relates to, among others,polynucleotides encoding the aforementioned fragments, polynucleotidesthat hybridize to polynucleotides encoding the fragments, particularlythose that hybridize under stringent conditions, and polynucleotides,such as PCR primers, for amplifying polynucleotides that encode thefragments. In these regards, preferred polynucleotides are those thatcorrespond to the preferred fragments, as discussed above.

Vectors, host cells, expression

The present invention also relates to vectors which containpolynucleotides of the present invention, host cells which aregenetically engineered with vectors of the invention and the productionof polypeptides of the invention by recombinant techniques.

Host cells can be genetically engineered to incorporate polynucleotidesand express polypeptides of the present invention. For instance,polynucleotides may be introduced into host cells using well knowntechniques of infection, transduction, transfection, transvection andtransformation. The polynucleotides may be introduced alone or withother polynucleotides. Such other polynucleotides may be introducedindependently, co-introduced or introduced joined to the polynucleotidesof the invention.

Thus, for instance, polynucleotides of the invention may be transfectedinto host cells with another, separate polynucleotide encoding aselectable marker, using standard techniques for co-transfection andselection in, for instance, mammalian cells. In this case thepolynucleotides generally will be stably incorporated into the host cellgenome.

Alternatively, the polynucleotides may be joined to a vector containinga selectable marker for propagation in a host. The vector construct maybe introduced into host cells by the aforementioned techniques.Generally, a plasmid vector is introduced as DNA in a precipitate, suchas a calcium phosphate precipitate, or in a complex with a chargedlipid. Electroporation may also be used to introduce polynucleotidesinto a host. If the vector is a virus, it may be packaged in vitro orintroduced into a packaging cell and the packaged virus may betransduced into cells. A wide variety of techniques suitable for makingpolynucleotides and for introducing polynucleotides into cells inaccordance with this aspect of the invention are well known and routineto those of skill in the art. Such techniques are reviewed at length inSambrook et al. which is illustrative of the many laboratory manualsthat detail these techniques.

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single- or double-stranded phage vector, ora single- or double-stranded RNA or DNA viral vector. Such vectors maybe introduced into cells as polynucleotides, preferably DNA, by wellknown techniques for introducing DNA and RNA into cells. The vectors, inthe case of phage and viral vectors may also be and preferably areintroduced into cells as packaged or encapsidated virus by well knowntechniques for infection and transduction. Viral vectors may bereplication competent or replication defective. In the latter case,viral propagation generally will occur only in complementing host cells.

Preferred among vectors, in certain respects, are those for expressionof polynucleotides and polypeptides of the present invention. Generally,such vectors comprise cis-acting control regions effective forexpression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors are either supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

In certain preferred embodiments in this regard, the vectors provide forspecific expression. Such specific expression may be inducibleexpression or expression only in certain types of cells or bothinducible and cell-specific expression. Particularly preferred amonginducible vectors are vectors that can be induced for expression byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives. A variety of vectors suitable to this aspect ofthe invention, including constitutive and inducible expression vectorsfor use in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art.

The engineered host cells can be cultured in conventional nutrientmedia, which may be modified as appropriate for, inter alia, activatingpromoters, selecting transformants or amplifying genes. Cultureconditions, such as temperature, pH and the like, previously used withthe host cell selected for expression, generally will be suitable forexpression of polypeptides of the present invention as will be apparentto those of skill in the art.

A great variety of expression vectors can be used to express apolypeptide of the invention. Such vectors include chromosomal, episomaland virus-derived vectors e.g., vectors derived from bacterial plasmids,bacteriophages, yeast episomes, yeast chromosomal elements, and virusessuch as baculoviruses, papova viruses, SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, cosmids and phagemids.Generally, any vector suitable to maintain, propagate or expresspolynucleotides to produce a polypeptide in a host may be used forexpression in this regard.

The appropriate DNA sequence may be inserted into the vector by any of avariety of well-known and routine techniques. In general, a DNA sequencefor expression is joined to an expression vector by cleaving the DNAsequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. Procedures for restriction and ligation that can be usedto this end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskilled in the art, are set forth in great detail in Sambrook et al.

The DNA sequence in the expression vector is operatively linked toappropriate expression control sequence(s), including, for instance, apromoter to direct MRNA transcription. Representatives of such promotersinclude the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name just a few of the well-known promoters. It will beunderstood that numerous other promoters useful in this aspect of theinvention are well known and may be routinely employed by those of skillin the manner illustrated by the discussion and the examples herein.

In general, expression constructs will contain sites for transcriptioninitiation and termination, and, in the transcribed region, a ribosomebinding site for translation. The coding portion of the maturetranscripts expressed by the constructs will include a translationinitiating AUG at the beginning and a termination codon appropriatelypositioned at the end of the polypeptide to be translated.

In addition, the constructs may contain control regions that regulate aswell as engender expression. Generally, in accordance with many commonlypracticed procedures, such regions will operate by controllingtranscription. Examples include repressor binding sites and enhancers,among others.

Vectors for propagation and expression generally will include selectablemarkers. Selectable marker genes provide a phenotypic trait forselection of transformed host cells. Preferred markers include, but arenot limited to, dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, and tetracycline or ampicillin resistance genesfor culturing E. coli and other bacteria. Such markers may also besuitable for amplification. Alternatively, the vectors may containadditional markers for this purpose.

The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable for expressiontherein of a desired polypeptide. Representative examples of appropriatehosts include bacterial cells, such as E coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Hosts of a greatvariety of expression constructs are well known, and those of skill willbe enabled by the present disclosure to routinely select a host forexpressing a polypeptide in accordance with this aspect of the presentinvention.

More particularly, the present invention also includes recombinantconstructs, such as expression constructs, comprising one or more of thesequences described above. The constructs comprise a vector, such as aplasmid or viral vector, into which such a sequence of the invention hasbeen inserted. The sequence may be inserted in a forward or reverseorientation. In certain preferred embodiments in this regard, theconstruct further comprises regulatory sequences, including, forexample, a promoter, operably linked to the sequence. Large numbers ofsuitable vectors and promoters are known to those of skill in the art,and there are many commercially available vectors suitable for use inthe present invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors preferred for use in bacteria are pQE70,pQE60 and pQE9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. These vectors are listed solely by way ofillustration of the many commercially available and well known vectorsthat are available to those of skill in the art for use in accordancewith this aspect of the present invention. It will be appreciated thatany other plasmid or vector suitable for, for example, introduction,maintenance, propagation or expression of a polynucleotide orpolypeptide of the invention in a host may be used in this aspect of theinvention.

Promoter regions can be selected from any desired gene using vectorsthat contain a reporter transcription unit lacking a promoter region,such as a chloramphenicol acetyl transferase ("CAT") transcription unit,downstream of a restriction site or sites for introducing a candidatepromoter fragment; i.e., a fragment that may contain a promoter. As iswell known, introduction into the vector of a promoter-containingfragment at the restriction site upstream of the CAT gene engendersproduction of CAT activity, which can be detected by standard CATassays. Vectors suitable to this end are well known and readilyavailable. Two examples of such vectors include pKK232-8 and pCM7. Thus,promoters for expression of polynucleotides of the present inventioninclude not only well known and readily available promoters, but alsopromoters that may be readily obtained by the foregoing technique, usinga reporter gene.

Among known bacterial promoters suitable for expression ofpolynucleotides and polypeptides in accordance with the presentinvention are the E. coli ladI and lacZ promoters, the T3 and T7promoters, the gpt promoter, the lambda PR, PL promoters and the trppromoter.

Among known eukaryotic promoters suitable in this regard are the CMVimmediate early promoter, the HSV thymidine kinase promoter, the earlyand late SV40 promoters, the promoters of retroviral LTRs, such as thoseof the Rous Sarcoma Virus("RSV"), and metallothionein promoters, such asthe mouse metallothionein-I promoter.

Selection of appropriate vectors and promoters for expression in a hostcell is a well known procedure and the requisite techniques forconstruction of expression vectors, introduction of the vector into thehost and expression in the host are routine skills in the art.

The present invention also relates to host cells containing theabove-described constructs. The host cell can be a higher eukaryoticcell, such as a mammalian cell, a lower eukaryotic cell, such as a yeastcell, or a prokaryotic cell, such as a bacterial cell.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals.

Constructs in host cells can be used in a conventional manner to producethe gene product encoded by the recombinant sequence. Alternatively, thepolypeptides of the invention can be synthetically produced byconventional peptide synthesizers.

Mature proteins can be expressed in mammalian cells, yeast, bacteria, orother cells under the control of appropriate promoters. Cell-freetranslation systems can also be employed to produce such proteins usingRNAs derived from the DNA constructs of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al.

Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells following exposureto the vector. Among suitable promoters are those derived from the genesthat encode glycolytic enzymes such as 3-phosphoglycerate kinase("PGK"), a-factor, acid phosphatase, and heat shock proteins, amongothers. Selectable markers include the ampicillin resistance gene of E.coli and the trpl gene of S. cerevisiae.

Transcription of DNA encoding the polypeptides of the present inventionby higher eukaryotes may be increased by inserting an enhancer sequenceinto the vector. Enhancers are cis-acting elements of DNA, usually fromabout 10 to 300 bp, that act to increase transcriptional activity of apromoter in a given host cell-type. Examples of enhancers include theSV40 enhancer, which is located on the late side of the replicationorigin at bp 100 to 270, the cytomegalovirus early promoter enhancer,the polyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

A polynucleotide of the invention encoding the heterologous structuralsequence of a polypeptide of the invention generally will be insertedinto the vector using standard techniques so that it is operably linkedto the promoter for expression. The polynucleotide will be positioned sothat the transcription start site is located appropriately 5' to aribosome binding site. The ribosome binding site will be 5' to the AUGthat initiates translation of the polypeptide to be expressed.Generally, there will be no other open reading frames that begin with aninitiation codon, usually AUG, and lie between the ribosome binding siteand the initiation codon. Also, generally, there will be a translationstop codon at the end of the polypeptide and a polyadenylation signaland transcription termination signal appropriately disposed at the 3'end of the transcribed region.

Appropriate secretion signals may be incorporated into the expressedpolypeptide for secretion of the translated protein into the lumen ofthe endoplasmic reticulum, the periplasmic space or the extracellularenvironment. The signals may be endogenous to the polypeptide orheterologous.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus, for example, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the polypeptide to improve stability andpersistence in the host cell during purification or subsequent handlingand storage. A region may also be added to the polypeptide to facilitatepurification. Such regions may be removed prior to final preparation ofthe polypeptide. The addition of peptide moieties to polypeptides toengender secretion or excretion, to improve stability and to facilitatepurification, among others are familiar and routine techniques in theart.

Suitable prokaryotic hosts for propagation, maintenance or expression ofpolynucleotides and polypeptides in accordance with the inventioninclude Escherichia coli, Bacillus subtilis and Salmonella typhimurium.Various species of Pseudomonas, Streptomyces, and Staphylococcus arealso suitable hosts in this regard. Moreover, many other hosts alsoknown to those of skill may be employed in this regard.

As a representative but non-limiting example, useful expression vectorsfor bacteria can comprise a selectable marker and bacterial origin ofreplication derived from commercially available plasmids comprisinggenetic elements of the well known cloning vector pBR322 (ATCC 37017).Such commercial vectors include, for example, pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, Wis.,USA). In these vectors, the pBR322 "backbone" sections are combined withan appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain, the host strain isgrown to an appropriate cell density. Where the selected promoter isinducible, it is induced by appropriate means (e.g., temperature shiftor exposure to chemical inducer) and cells are cultured for anadditional period. Cells typically then are harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

Microbial cells employed in expression of proteins can be disrupted byany convenient method, including freeze-thaw cycling, sonication,mechanical disruption, or use of cell lysing agents. Such methods arewell known to those skilled in the art.

Various mammalian cell culture systems can be employed for expression,as well. Examples of mammalian expression systems include the C127, 3T3,CHO, HeLa, human kidney 293 and BHK cell lines and the COS-7 line ofmonkey kidney fibroblasts described by Gluzman et al., Cell, 1981, 23:175.

Mammalian expression vectors comprise an origin of replication, asuitable promoter and enhancer, and any necessary ribosome bindingsites, polyadenylation sites, splice donor and acceptor sites,transcriptional termination sequences, and 5' flanking non-transcribedsequences that are necessary for expression. In certain preferredembodiments, DNA sequences derived from the SV40 splice sites and theSV40 polyadenylation sites are used for required non-transcribed geneticelements.

The ELF3 polypeptide can be recovered and purified from recombinant cellcultures by well known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography("HPLC") is employed for purification. Well known techniques forrefolding proteins may be employed to regenerate active conformationwhen the polypeptide is denatured during isolation and or purification.

Polypeptides of the present invention include naturally purifiedpolypeptides, polypeptides produced by chemical synthetic procedures,and polypeptides produced by recombinant techniques from a prokaryoticor eukaryotic host, including, for example, bacterial, yeast, higherplant, insect and mammalian cells. Depending upon the host employed in arecombinant production procedure, the polypeptides of the presentinvention may be glycosylated or non-glycosylated. In addition,polypeptides of the invention may include an initial modified methionineresidue, in some cases as a result of host-mediated processes.

ELF3 polynucleotides and polypeptides may be used in accordance with thepresent invention for a variety of applications, particularly those thatmake use of the chemical and biological properties of ELF3. Additionalapplications relate to diagnosis and to treatment of disorders of cells,tissues and organisms. These aspects of the invention are illustratedfurther by the following discussion.

Polynucleotide assays

This invention is also related to the use of ELF3 polynucleotides todetect complementary polynucleotides for use, for example, as adiagnostic reagent. Detection of a mutated form of ELF3 associated witha dysfunction will provide a diagnostic tool that can add to or definediagnosis of a disease or susceptibility to a disease which results fromunder-expression, over-expression or altered expression of ELF3.Individuals carrying mutations in the human ELF3 gene may be detected atthe DNA level by a variety of techniques. Nucleic acids for diagnosismay be obtained from a patient's cells, such as from blood, urine,saliva, tissue biopsy or autopsy material. The genomic DNA may be useddirectly for detection or may be amplified enzymatically by using PCRprior to analysis (Saiki et al., Nature, 1986, 324: 163-166). RNA orcDNA may also be used in similar fashion. As an example, PCR primerscomplementary to the nucleic acid encoding ELF3 can be used to identifyand analyze ELF3 expression and mutations. For example, deletions andinsertions can be detected by a change in size of the amplified productin comparison to the normal genotype. Point mutations can be identifiedby hybridizing amplified DNA to radiolabeled ELF3 RNA or radiolabeledELF3 antisense DNA sequences. Perfectly matched sequences can bedistinguished from mismatched duplexes by RNase A digestion or bydifferences in melting temperatures.

Sequence differences between a reference gene and genes having mutationsmay also be revealed by direct DNA sequencing. In addition, cloned DNAsegments may be employed as probes to detect specific DNA segments. Thesensitivity of such methods can be greatly enhanced by appropriate useof PCR or other amplification methods. For example, a sequencing primeris used with double-stranded PCR product or a single-stranded templatemolecule generated by a modified PCR. The sequence determination isperformed by conventional procedures with radiolabeled nucleotide or byautomatic sequencing procedures with fluorescent-tags.

Genetic testing based on DNA sequence differences may be achieved bydetection of alterations in electrophoretic mobility of DNA fragments ingels, with or without denaturing agents. Small sequence deletions andinsertions can be visualized by high resolution gel electrophoresis. DNAfragments of different sequences may be distinguished on denaturingformamide gradient gels in which the mobilities of different DNAfragments are retarded in the gel at different positions according totheir specific melting or partial melting temperatures (see, e.g., Myerset al., Science, 1985, 230: 1242).

Sequence changes at specific locations may also be revealed by nucleaseprotection assays, such as RNase and S1 protection or the chemicalcleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA, 1985,85: 4397-4401).

Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms ("RFLP") and Southern blottingof genomic DNA.

In accordance with a further aspect of the invention, there is provideda process for diagnosing or determining a susceptibility to cancer, inparticular, prostate, breast, lung and other epithelial tumors, amongothers. Specific chromosomal translocations or aberrant transcripts ofELF3 may also be usefull in the diagnosis of these tumors, among others;and the nucleic acid sequences described above may be employed in anassay for ascertaining such susceptibility. Thus, for example, the assaymay be employed to determine the presence of the human ELF3 gene, or achromosomal translocation or aberrant transcript as herein described,which is indicative of a susceptibility to cancer, and in particular,prostate, breast, lung and other epithelial tumors, among others.

The invention provides a process for diagnosing diseases, particularly,cancers such as prostate, breast, lung and other epithelial tumors,among others; comprising determining from a sample derived from apatient an abnormally decreased or increased level of expression ofpolynucleotide having the sequence of FIG. 1, SEQ ID NO: 1. Decreased orincreased expression of polynucleotide can be measured using any of themethods well known in the art for the quantitation of polynucleotides,such as, for example, PCR, RT-PCR, RNase protection, Northern blottingand other hybridization methods.

In addition to more conventional gel-electrophoresis and DNA sequencing,mutations can also be detected by in situ analysis.

Chromosome assays

The sequences of the present invention are also valuable for chromosomeidentification. The sequence is specifically targeted to and canhybridize with a particular location on an individual human chromosome.Moreover, there is a current need for identifying particular sites onthe chromosome. Few chromosome marking reagents based on actual sequencedata (repeat polymorphisms) are presently available for markingchromosomal location. The mapping of DNAs to chromosomes according tothe present invention is an important first step in correlating thosesequences with gene associated disease.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the cDNA. Computer analysis of the 3'untranslated region is used to rapidly select primers that do not spanmore than one exon in the genomic DNA, because primers that span morethan one exon could complicate the amplification process. These primersare then used for PCR screening of somatic cell hybrids containingindividual human chromosomes. Only those hybrids containing the humangene corresponding to the primer will yield an amplified fragment.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular DNA to a particular chromosome. Using the present inventionwith the same oligonucleotide primers, sublocalization can be achievedwith panels of fragments from specific chromosomes or pools of largegenomic clones in an analogous manner. Other mapping strategies that canbe used similarly to map to the chromosome include in situhybridization, prescreening with labeled flow-sorted chromosomes andpreselection by hybridization to construct chromosome specific-cDNAlibraries.

Fluorescence in situ hybridization (FISH) of a cDNA clone to a metaphasechromosomal spread can be used to provide a precise chromosomal locationin one step. This technique can be used with cDNAs as short as 50 to 60bases. For a review of this technique, see Verma et al., HUMANCHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon press, New York,1988.

As an example of how this technique is performed, ELF3 DNA is digestedand purified with a QIAEX II DNA purification kit (Qiagen, Inc.,Chatsworth, Calif.) and ligated to a Super Cosl cosmid vector(Stratagene, La Jolla, Calif.). DNA is purified using a Qiagen PlasmidPurification Kit (Qiagen Inc., Chatsworth, Calif.) and 1 mg is labeledby nick translation in the presence of Biotin-dATP using a BioNickLabeling Kit (GibcoBRL, Life Technologies Inc., Gaithersburg, Md.).Biotinylation is detected with GENE-TECT Detection System (ClontechLaboratories, Inc. Palo Alto, Calif.). In situ hybridization isperformed on slides using ONCOR Light Hybridization Kit (Oncor,Gaithersburg, Md.) to detect single copy sequences on metaphasechromosomes. Peripheral blood of normal donors is cultured for threedays in RPMI 1640 supplemented with 20% FCS, 3% PHA andpenicillin/streptomycin, synchronized with 10⁻⁷ M methotrexate for 17hours, and washed twice with unsupplemented RPMI. Cells are thenincubated with 10⁻³ M thymidine for 7 hours. The cells are arrested inmetaphase after a 20 minute incubation with colcemid (0.5 μg/ml)followed by hypotonic lysis in 75 mM KCl for 15 minutes at 37° C. Cellpellets are then spun out and fixed in Carnoy's fixative (3:1methanol:acetic acid).

Metaphase spreads are prepared by adding a drop of the suspension ontoslides and air drying the suspension. Hybridization is performed byadding 100 ng of probe suspended in 10 ml of hybridization mix (50%formamide, 2xSSC, 1% dextran sulfate) with blocking human placental DNA(1 μg/ml). Probe mixture is denatured for 10 minutes in a 70° C. waterbath and incubated for 1 hour at 37° C., before placement on a prewarmed(37° C.) slide, previously denatured in 70% formamide/2xSSC at 70° C.,dehydrated in ethanol series, and chilled to 4° C.

Slides are incubated for 16 hours at 37° C. in a humidified chamber.Slides are washed in 50% formamide/2xSSC for 10 minutes at 41° C. and2xSSC for 7 minutes at 37° C. Hybridization probe is detected byincubation of the slides with FITC-Avidin (Oncor, Gaithersburg, Md.),according to the manufacturer's protocol. Chromosomes are counterstainedwith propridium iodine suspended in mounting medium. Slides arevisualized using a Leitz ORTHOPLAN 2-epifluorescence microscope and fivecomputer images are taken using a Imagenetics Computer and MacIntoshprinter.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance in Man (available on line through Johns HopkinsUniversity Welch Medical Library). The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

It is then necessary to determine the differences in the cDNA or genomicsequence between affected and unaffected individuals. If a mutation isobserved in some or all of the affected individuals but not in anynormal individuals, then the mutation is likely to be the causativeagent of the disease.

With current resolution of physical mapping and genetic mappingtechniques, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of between 50 and 500 potentialcausative genes assuming 1 megabase mapping resolution and one gene per20 kb.

Polypeptide assays

The present invention also relates to diagnostic assays for detectinglevels of ELF3 protein in cells and tissues. Such assays may bequantitative or qualitative, Thus, for instance, a diagnostic assay inaccordance with the invention for detecting over-expression of ELF3protein compared to normal control tissue samples may be used to detectthe presence of a disease or disorder such as cancer, and in particularprostate, breast, lung or other epithelial tumors, among others. Assaytechniques that can be used to determine levels of a protein, such as anELF3 protein of the present invention, in a sample derived from a hostare well-known to those of skill in the art. Such assay methods includeradioimmuno assays, competitive-binding assays, Western Blot analysisand enzyme linked immunosorbent assays (ELISAs). Among these, ELISAs arefrequently preferred. An ELISA assay initially comprises preparing anantibody specific to ELF3, preferably a monoclonal antibody. In additiona reporter antibody generally is prepared which binds to the monoclonalantibody. The reporter antibody is attached to a detectable reagent suchas a radioactive, fluorescent or enzymatic reagent, in this example,horseradish peroxidase enzyme.

To carry out an ELISA, a sample is removed from a host and incubated ona solid support, e.g. a polystyrene dish, that binds the proteins in thesample. Any free protein binding sites on the dish are then covered byincubating with a non-specific protein such as bovine serum albumin. Themonoclonal antibody is then incubated in the dish during which time themonoclonal antibodies attach to any ELF3 proteins attached to thepolystyrene dish. Unbound monoclonal antibody is washed out with buffer.The reporter antibody linked to horseradish peroxidase is placed in thedish resulting in binding of the reporter antibody to any monoclonalantibody bound to ELF3. Unattached reporter antibody is then washed out.Reagents for peroxidase activity, including a calorimetric substrate arethen added to the dish. Immobilized peroxidase, linked to ELF3 throughthe primary and secondary antibodies, produces a colored reactionproduct. The amount of color developed in a given time period indicatesthe amount of ELF3 protein present in the sample. Quantitative resultstypically are obtained by reference to a standard curve.

A competition assay may also be employed wherein antibodies specific toELF3 attached to a solid support and labeled ELF3 and a sample derivedfrom the host are passed over the solid support. The amount of detectedlabel attached to the solid support can be correlated to a quantity ofELF3 in the sample.

Antibodies

The polypeptides, their fragments or other derivatives, or analogsthereof, or cells expressing them can also be used as immunogens toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of a Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptide itself. In this manner, even a sequence encodingonly a fragment of the polypeptide can be used to generate antibodiesbinding the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler, G. and Milstein, C.,Nature, 1975, 256: 495-497), the trioma technique, the human B-cellhybridoma technique (Kozbor et al, Immunology Today, 1983, 4: 72) andthe EBV-hybridoma technique (Cole et al., MONOCLONAL ANTIBODIES ANDCANCER THERAPY, pages 77-96, Alan R. Liss, Inc., 1985).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can also be adapted to produce single chainantibodies to immunogenic polypeptide products of this invention. Also,transgenic mice, or other organisms including other mammals, may be usedto express humanized antibodies to immunogenic polypeptide products ofthis invention.

The above-described antibodies may be employed to isolate or to identifyclones expressing the polypeptide or purify the polypeptide of thepresent invention by attachment of the antibody to a solid support forisolation and/or purification by affinity chromatography.

Antibodies against ELF3 may also be employed to inhibit tumor formation,in particular prostate, breast, lung or other epithelial tumors, amongothers. ELF3 antibodies can also be used on nuclear lysates of diseasedtissue to determine whether or not the binding ability of ELF3 ismodulated as compared to normal tissue. In addition, ELF3 antibodies canbe used with genomic DNA to identify promoters of genes to which ELF3binds.

ELF3 binding molecules and assays

ELF3 can be used to isolate proteins which interact with it; thisinteraction can be a target for interference. Inhibitors ofprotein-protein interactions between ELF3 and other factors could leadto the development of pharmaceutical agents for the modulation of ELF3activity.

Thus, this invention also provides a method for identification ofbinding molecules to ELF3. Genes encoding proteins for binding moleculesto ELF3 can be identified by numerous methods known to those of skill inthe art, for example, ligand panning and FACS sorting. Such methods aredescribed in many laboratory manuals such as Coligan et al, CURRENTPROTOCOLS IN IMMUNOLOGY 1, Chapter 5, 1991.

For example, the yeast two-hybrid system provides methods for detectingthe interaction between a first test protein and a second test protein,in vivo, using reconstitution of the activity of a transcriptionalactivator. The method is disclosed in U.S. Pat. No. 5,283,173; reagentsare available from Clontech and Stratagene. Briefly, ELF3 cDNA is fusedto a Ga.4 transcription factor DNA binding domain and expressed in yeastcells. cDNA library members obtained from cells of interest are fused toa transactivation domain of Gal4. cDNA clones which express proteinswhich can interact with ELF3 will lead to reconstitution of Ga14activity and transactivation of expression of a reporter gene such asGal4-lacZ.

An alternative method involves screening of λgt11, λZAP (Stratagene) orequivalent cDNA expression libraries with recombinant ELF3. RecombinantELF3 protein or fragments thereof are fused to small peptide tags suchas FLAG, HSV or GST. The peptide tags can possess convenientphosphorylation sites for a kinase such as heart muscle creatine kinaseor they can be biotinylated. Recombinant ELF3 can be phosphorylated with³² P! or used unlabeled and detected with streptavidin or antibodiesagainst the tags. λgt11cDNA expression libraries are made from cells ofinterest and are incubated with the recombinant ELF3, washed and cDNAclones which interact with ELF3 isolated. Such methods are routinelyused by skilled artisans. See, e.g., Sambrook et al.

Another method is the screening of a mammalian expression library. Inthis method, cDNAs are cloned into a vector between a mammalian promoterand polyadenylation site and transiently transfected in COS or 293cells. Forty-eight hours later, the binding protein is detected byincubation of fixed and washed cells with labeled BLF3. In a preferredembodiment, the ELF3 is iodinated, and any bound ELF3 is detected byautoradiography. See Sims et al., Science, 1988, 241: 585-589 andMcMahan et al., EMBO J., 1991, 10: 2821-2832. In this manner, pools ofcDNAs containing the cDNA encoding the binding protein of interest canbe selected and the cDNA of interest can be isolated by furthersubdivision of each pool followed by cycles of transient transfection,binding and autoradiography. Alternatively, the cDNA of interest can beisolated by transfecting the entire cDNA library into mammalian cellsand panning the cells on a dish containing ELF3 bound to the plate.Cells which attach after washing are lysed and the plasmid DNA isolated,amplified in bacteria, and the cycle of transfection and panningrepeated until a single cDNA clone is obtained. See Seed et al, Proc.Natl. Acad. Sci. USA, 1987, 84: 3365 and Aruffo et al., EMBO J, 1987, 6:3313. If the binding protein is secreted, its cDNA can be obtained by asimilar pooling strategy once a binding or neutralizing assay has beenestablished for assaying supernatants from transiently transfectedcells. General methods for screening supernatants are disclosed in Wonget al., Science, 1985, 228: 810-815.

Another method involves isolation of proteins interacting with ELF3directly from cells. Fusion proteins of ELF3 with GST or small peptidetags are made and immobilized on beads. Biosynthetically labeled orunlabeled protein extracts from the cells of interest are prepared,incubated with the beads and washed with buffer. Proteins interactingwith ELF3 are eluted specifically from the beads and analyzed bySDS-PAGE. Binding partner primary amino acid sequence data are obtainedby microsequencing. Optionally, the cells can be treated with agentsthat induce a functional response such as tyrosine phosphorylation ofcellular proteins. An example of such an agent would be a growth factoror cytokine such as interleukin-2.

Another method is immunoaffinity purification. Recombinant ELF3 isincubated with labeled or unlabeled cell extracts and immunoprecipitatedwith anti-ELF3 antibodies. The immunoprecipitate is recovered withprotein A-Sepharose and analyzed by SDS-PAGE. Unlabelled proteins arelabeled by biotinylation and detected on SDS gels with streptavidin.Binding partner proteins are analyzed by microsequencing. Further,standard biochemical purification steps known to those skilled in theart may be used prior to microsequencing.

Yet another alternative method involves screening of peptide librariesfor binding partners. Recombinant tagged or labeled ELF3 is used toselect peptides from a peptide or phosphopeptide library which interactwith ELF3. Sequencing of the peptides leads to identification ofconsensus peptide sequences which might be found in interactingproteins.

ELF3 binding partners identified by any of these methods or othermethods, which would be known to those of ordinary skill in the art, aswell as those putative binding partners discussed above, can be used inthe assay method of the invention. Assaying for the presence ofELF3/binding partner complex is accomplished by, for example, the yeasttwo-hybrid system, ELISA or immunoassays using antibodies specific forthe complex. In the presence of test substances which interrupt orinhibit formation of ELF3/binding partner interaction, a decreasedamount of complex will be determined relative to a control lacking thetest substance.

Assays for free ELF3 or binding partner are accomplished by, forexample, ELISA or immunoassay using specific antibodies or by incubationof radiolabeled ELF3 with cells or cell membranes followed bycentrifugation or filter separation steps. In the presence of testsubstances which interrupt or inhibit formation of ELF3/binding partnerinteraction, an increased amount of free ELF3 or free binding partnerwill be determined relative to a control lacking the test substance.

Polypeptides of the invention can also be used to assess ELF3 bindingcapacity of ELF3 binding molecules in cells or in cell-freepreparations.

Agonists and antagonists--assays and molecules

Transcription factors such as ELF3 elicit their biological andpathological responses by activating or repressing genes that theyregulate. These transcription factors may be activated or inhibited atseveral steps in the pathway, for example, from ligands that signalligand/receptor interactions through these molecules, to receptors thatmay be activated, to secondary messengers that are activated, toposttranslational modifications of the transcription factors, tocompounds that interact with it, or to compounds that directly affectits activity. The EIF3 of the present invention may be employed in ascreening process for compounds which activate (agonists) or inhibitactivation (antagonists) of this transcription factor and/or the genesthat it regulates.

In general, such screening procedures involve producing appropriatecells which express polypeptide of the present invention. Such cellsinclude cells from mammals, yeast, Drosophila or E. coli. In particular,a polynucleotide encoding the polypeptide of the present invention isemployed to transfect cells to thereby express the ELF3. Cellsexpressing the polypeptide are then contacted with a test compound toobserve binding, stimulation or inhibition of a functional response.

One such screening procedure involves the use of melanophores which aretransfected to express the ELF3 of the present invention. Such ascreening technique is described in PCT WO92/01810. In one embodiment,this technique is employed to screen for compounds which inhibitactivation of the polypeptide of the present invention by contacting themelanophore cells which encode the polypeptide with both the ELF3binding molecule and a compound to be screened. Inhibition of the signalgenerated by the ELF3 binding molecule indicates that a compound is apotential antagonist for the ELF3, i.e., inhibits activation of thepolypeptide. The technique may also be employed for screening ofcompounds which activate the polypeptide by contacting such cells withcompounds to be screened and determining whether such compound generatesa signal, i.e., activates the polypeptide.

Other screening techniques include the use of cells which express theELF3 (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by activation of the polypeptide. (Seee.g., Science, 1989, 246: 181-296). In this technique, compounds may becontacted with cells expressing the polypeptide of the presentinvention. A second messenger response, e.g., signal transduction or pHchange, is then measured to determine whether the potential compoundactivates or inhibits the polypeptide.

Another screening technique involves introducing RNA encoding the ELF3into Xenopus oocytes to transiently express the polypeptide. The oocytesare then contacted with the ELF3 binding molecules and a compound to bescreened. Inhibition or activation of the polypeptide is then determinedby detection of a signal, such as, calcium, proton, or other ions, inthe case of screening for compounds which are thought to inhibitactivation of the polypeptide.

Another screening technique involves expressing ELF3 linked tophospholipase C or D. Representative examples of such cells include, butare not limited to, endothelial cells, smooth muscle cells, andembryonic kidney cells. The screening may be accomplished as hereinabovedescribed by detecting activation of ELF3 or inhibition of activation ofthe ELF3 from the phospholipase second signal.

Another method involves screening for compounds which are antagonistsand thus inhibit activation of the polypeptide of the present inventionby determining inhibition of binding of labeled ligand to cells whichhave the polypeptide on the surface thereof. Such a method involvestransfecting a eukaryotic cell with DNA encoding ELF3 such that the cellexpresses the polypeptide on its surface. The cells are then contactedwith a compound in the presence of a labeled form of a known ligand. Theligand can be labeled, e.g., by radioactivity. The amount of labeledligand bound to the polypeptide is measured, e.g., by measuringradioactivity associated with transfected cells or membrane from thesecells. If the compound binds to the ELF3, the binding of labeled ligandto the polypeptide is inhibited as determined by a reduction of boundlabeled ligand.

Another method involves screening for ELF3 inhibitors by determininginhibition or stimulation of ELF3-mediated cAMP and/or adenylate cyclaseaccumulation. Such a method involves transfecting a eukaryotic cell withELF3 to express the polypeptide on the cell surface. The cell is thenexposed to potential antagonists in the presence of ELF3. The amount ofcAMP accumulation is then measured. If the potential antagonist bindsthe polypeptide, and thus inhibits ELF3 binding, the levels ofELF3-mediated cAMP, or adenylate cyclase, activity will be reduced orincreased.

Other methods for detecting agonists or antagonists for the polypeptideof the present invention include the yeast based technologies asdescribed in U.S. Pat. No. 5,482,835.

The present invention also provides a method for determining whether aligand not known to be capable of binding to an ELF3 can bind to suchpolypeptide. This method comprises contacting a mammalian cell whichexpresses an ELF3 with the ligand under conditions permitting binding ofligands to the ELF3, and detecting the presence of a ligand which bindsto the polypeptide thereby determining whether the ligand binds to theELF3. The systems hereinabove described for determining agonists and/orantagonists may also be employed for determining ligands which bind tothe ELF3.

Examples of potential ELF3 polypeptide antagonists include antibodiesor, in some cases, oligonucleotides which bind to the polypeptide but donot elicit a second messenger response such that the activity of thepolypeptide is prevented.

Potential antagonists also include proteins which are closely related toELF3, i.e. a fragment of ELF3, which have lost biological function and,when binding to a receptor for ELF3, elicit no response.

A potential antagonist also includes an antisense construct preparedthrough the use of antisense technology. Antisense technology can beused to control gene expression through triple-helix formation orantisense DNA or RNA, both methods of which are based on binding of apolynucleotide to DNA or RNA. For example, the 5' coding portion of thepolynucleotide sequence, which encodes for the mature polypeptides ofthe present invention, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix -see Lee et al., Nucl AcidsRes., 1979, 6: 3073; Cooney et al., Science, 1988, 241: 456; and Dervanet al., Science, 1991, 251: 1360), thereby preventing transcription andproduction of the ELF3 polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the ELF3 polypeptide (antisense--see Okano, J Neurochem.,(1991) 56: 560; Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988)). The oligonucleotidesdescribed above can also be delivered to cells such that the antisenseRNA or DNA is expressed in vivo to inhibit production of the ELF3polypeptide.

Another potential antagonist is a small molecule which binds to the ELF3polypeptide, making it inaccessible to ligands such that normalbiological activity is prevented. Examples of small molecules include,but are not limited to, small peptides or peptide-like molecules.

Alternatively, antagonists or agonists of the present invention maycomprise molecules which activate or repress genes regulated by thistranscription factor. Electrophoretic mobility shift assays where ELF3binding sites in promoters are used together with ELF3 recombinantproteins can be used to identify genes regulated by ELF3. Modulation ofthe expression of these genes by test compounds to identify potentialantagonists and agonist can then performed in accordance with the abovedescribed methods.

ELF3 proteins are ubiquitous in the mammalian host and are responsiblefor many biological functions, including many pathologies. Accordingly,it is desirous to find compounds and drugs which can inhibit thefunction of ELF3 or genes regulated thereby.

Antagonists for ELF3 may be employed for a variety of therapeutic andprophylactic purposes for such diseases or disorders as cancer, and inparticular prostate, breast, lung and other epithelial tumors, amongothers.

This invention additionally provides a method of treating an abnormalcondition related to an excess of ELF3 activity which comprisesadministering to a subject an inhibitor compound (antagonist) ashereinabove described along with a pharmaceutically acceptable carrierin an amount effective to inhibit expression and/or activation of theELF3 protein, or by inhibiting a second signal, and thereby alleviatingthe abnormal condition. By pharmaceutically acceptable carrier, it ismeant to include, but is not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol, and combinations thereof. Theformulation should suit the mode of administration. Selection of anappropriate carrier in accordance with the mode of administration isroutinely performed by those skilled in the art.

The invention further relates to pharmaceutical packs and kitscomprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.

Administration

Polypeptides and other compounds of the present invention may beemployed alone or in conjunction with other compounds, such astherapeutic compounds.

The pharmaceutical compositions may be administered in any effective,convenient manner including, for instance, administration by topical,oral, anal, vaginal, intravenous, intraperitoneal, intramuscular,subcutaneous, intranasal or intradermal routes, among others.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. In general, the compositions are administered in an amountof at least about 10 μg/kg body weight. In most cases they will beadministered in an amount not in excess of about 8 mg/kg body weight perday. Preferably, in most cases, the administered dose is from about 10μg/kg to about 1 mg/kg body weight, daily. It will be appreciated thatoptimum dosage will be determined by standard methods for each treatmentmodality and indication, taking into account the indication, itsseverity, route of administration, complicating conditions and the like.

Gene therapy

The ELF3 polynucleotides, polypeptides, agonists and antagonists thatare polypeptides may be employed in accordance with the presentinvention by expression of such polypeptides in treatment modalitiesoften referred to as "gene therapy."

Thus, for example, cells from a patient may be engineered with apolynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo.The engineered cells can then be provided to a patient to be treatedwith the polypeptide. In this embodiment, cells may be engineered exvivo, for example, by the use of a retroviral plasmid vector containingRNA encoding a polypeptide of the present invention. Such methods arewell-known in the art and their use in the present invention will beapparent from the teachings herein.

Similarly, cells may be engineered in vivo for expression of apolypeptide in vivo by procedures known in the art. For example, apolynucleotide of the invention may be engineered for expression in areplication defective retroviral vector, as discussed above. Theretroviral expression construct may then be isolated and introduced intoa packaging cell transduced with a retroviral plasmid vector containingRNA encoding a polypeptide of the present invention such that thepackaging cell now produces infectious viral particles containing thegene of interest. These producer cells may be administered to a patientfor engineering cells in vivo and expression of the polypeptide in vivo.These and other methods for administering a polypeptide of the presentinvention should be apparent to those skilled in the art from theteachings of the present invention.

Retroviruses from which the retroviral plasmid vectors herein abovementioned may be derived include, but are not limited to, Moloney MurineLeukemia Virus, Spleen Necrosis Virus, Rous Sarcoma Virus, HarveySarcoma Virus, Avian Leukosis Virus, Gibbon Ape Leukemia Virus, HumanImmunodeficiency Virus, Adenovirus, Myeloproliferative Sarcoma Virus,and Mammary Tumor Virus. In a preferred embodiment, the retroviralplasmid vector is derived from Moloney Murine Leukemia Virus.

Such vectors will include one or more promoters for expressing thepolypeptide. Suitable promoters which may be employed include, but arenot limited to, the retroviral LTR; the SV40 promoter; and the humancytomegalovirus (CMV) promoter described in Miller et al.,Biotechniques, 1989, 7: 980-990. Cellular promoters such as eukaryoticcellular promoters including, but not limited to, the histone, RNApolymerase III, and β-actin promoters can also be used. Additional viralpromoters which may be employed include, but are not limited to,adenovirus promoters, thymidine kinase (TK) promoters, and B19parvovirus promoters. The selection of a suitable promoter will beapparent to those skilled in the art from the teachings containedherein.

The nucleic acid sequence encoding the polypeptide of the presentinvention will be placed under the control of a suitable promoter.Suitable promoters which may be employed include, but are not limitedto, adenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs (including the modified retroviral LTRs hereinabove described); the β-actin promoter; and human growth hormonepromoters. The promoter may also be the native promoter which controlsthe gene encoding the polypeptide.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, Y-2,Y-AM, PA12, T19-14X, VT19-17-1H2, YCRE, YCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, A., Human Gene Therapy, 1990, 1:5-14. The vector may be transduced into the packaging cells through anymeans known in the art. Such means include, but are not limited to,electroporation, the use of liposomes, and CaPO₄ precipitation. In onealternative, the retroviral plasmid vector may be encapsulated into aliposome, or coupled to a lipid, and then administered to a host.

The producer cell line will generate infectious retroviral vectorparticles, which include the nucleic acid sequence(s) encoding thepolypeptides. Such retroviral vector particles may then be employed totransduce eukaryotic cells, either in vitro or in vivo. The transducedeukaryotic cells will express the nucleic acid sequence(s) encoding thepolypeptide. Eukaryotic cells which may be transduced include, but arenot limited to, embryonic stem cells, embryonic carcinoma cells, as wellas hematopoietic stem cells, hepatocytes, fibroblasts, myoblasts,keratinocytes, endothelial cells, and bronchial epithelial cells.

EXAMPLES

The present invention is further described by the following examples.The examples are provided solely to illustrate the invention byreference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

Certain terms used herein are explained in the foregoing glossary.

All examples are carried out using standard techniques, which are wellknown and routine to those of skill in the art, except where otherwisedescribed in detail. Routine molecular biology techniques of thefollowing examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al.

All parts or amounts set out in the following examples are by weight,unless otherwise specified.

Unless otherwise stated size separation of fragments in the examplesbelow is carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis ("PAGE") as described in Sambrook andnumerous other references such as Goeddel et al., Nucleic Acids Res.,1980, 8: 4057.

Unless described otherwise, ligations are accomplished using standardbuffers, incubation temperatures and times, approximately equimolaramounts of the DNA fragments to be ligated and approximately 10 units ofT4 DNA ligase ("ligase") per 0.5 μg of DNA.

Example 1

Expression and purification of human ELF3 using bacteria

The DNA sequence encoding human ELF3 in the deposited polynucleotide isamplified using PCR oligonucleotide primers specific to the amino acidcarboxyl terminal sequence of human ELF3 protein and to vector sequences3' to the gene. Additional nucleotides containing restriction sites tofacilitate cloning are added to the 5' and 3' sequences respectively.

The 5' oligonucleotide primer has the sequence:5'-CGGGATCCGCTGCAACCTGTGAGATTAGC-3' (SEQ ID NO: 3) containing theunderlined BamHI restriction site, which encodes codon 2 followed by 21nucleotides of the human ELF3 coding sequence set out in FIG. 1beginning with nucleotide 118 of codon 2.

The 3' primer has the sequence: 5'-GCAGATCTCAGTTCCGACTCTGGAGAACC-3' (SEQID NO: 4) containing the underlined BglII restriction site followed by21 nucleotides complementary to the last 22 nucleotides of the ELF3coding sequence set out in FIG. 1, including the stop codon.

The restrictions sites are convenient to restriction enzyme sites in thebacterial expression vectors pQE-30 (Qiagen, Inc. Chatsworth, Calif.)which are used for bacterial expression in these examples. pQE-30encodes ampicillin antibiotic resistance ("Ampr") and contains abacterial origin of replication ("ori"), an IPTG inducible promoter, aribosome binding site ("RBS"), a 6-His tag and restriction enzyme sites.

The amplified human ELF3 DNA is digested with BamHl and BglII. Thevector pQE-30 are digested with BamHI. The digested DNAs are thenligated together. Insertion of the ELF3 DNA into the BamHI siterestricted vector places the ELF3 coding region downstream of andoperably linked to the vector's IPTG-inducible promoter and in-framewith an initiating AUG appropriately positioned for translation of ELF3.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures. Such procedures are described in Sambrook. E. colistrain M15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses lac repressor and confers kanamycin resistance ("Kan^(r) "),is used in carrying out the illustrative example described here. Thisstrain, which is only one of many that are suitable for expressing ELF3,is available commercially from Qiagen. Transformants are identified bytheir ability to grow on LB plates in the presence of ampicillin.Plasmid DNA is isolated from resistant colonies and the identity of thecloned DNA is confirmed by restriction analysis.

Clones containing the desired constructs are grown overnight ("O/N") inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml).

The O/N culture is used to inoculate a large culture, at a dilution ofapproximately 1:100 to 1:250. The cells are grown to an optical densityat 600 nm ("OD⁶⁰⁰ ") of between 0.4 and 0.6.Isopropyl-B-D-thiogalactopyranoside ("IPTG") is then added to a finalconcentration of 1 mM to induce transcription from lac repressorsensitive promoters, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells are thenharvested by centrifugation and disrupted, by standard methods.Inclusion bodies are purified from the disrupted cells using routinecollection techniques, and protein is solubilized from the inclusionbodies in 6M guanidine. The 6M guanidine solution is added to TALONmatrix (Clontech, Palo Alto, Calif.) which binds ELF3 via the histidinetag at the N-terminal of the protein. The TALON Matrix is washed withbuffer containing 8M urea and 20 mM imidazole and eluted with buffercontaining 8M urea and 50 mM imidazole. The eluted protein is thedialyzed against dialysis buffer containing 20 mM HEPES buffer and 20%glycerol, thereby removing urea and refolding the protein.

Analysis of the preparation by standard methods of polyacrylamide gelelectrophoresis revealed that the preparation contained about 90%monomer ELF3 having the expected molecular weight of, approximately, 42kDa.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 4                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1920                                                              (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (iv) ANTI-SENSE: No                                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:                                      GCCGGGTAGGGGAGCGCAGCGGCCAGATACCTCAGCGCTACCTGGCGGAA50                          CTGGATTTCTCTCCCGCCTGCCGGCCTGCCTGCCACAGCCGGACTCCGCC100                         ACCCCGGTAGCCTCATGGCTGCAACCTGTGAGATTAGCAACATTTTTAGC150                         AACTACTTCAGTGCGATGTACAGCTCGGAGGACTCCACCTTGGCCTCTGT200                         TCCCCCTGCTGCCACCTTTGGGGCCGATGACTTGGTACTGACCCTGAGCA250                         ACCCCCAGATGTCATTGGAGGGTACAGAGAAGGCTAGCTGGTTGGGGGAA300                         CAGCCCCAGTTCTGGTCGAAGACGCAGGTTCTGGACTGGATCAGCTACCA350                         AGTGGAGAAGAACAAGTACGACGCAAGCGCCATTGACTTCTCACGATGTG400                         ACATGGATGGGGCCACCCTCTGCAATTGTGCCCTTGAGGAGCTGCGTCTG450                         GTCTTTGGGCCTCTGGGGGACCAACTCCATGCCCAGCTGCGAGACCTCAC500                         TTCCAGCTCTTCTGATGAGCTCAGTTGGATCATTGAGCTGCTGGAGAAGG550                         ATGGCATGGCCTTCCAGGAGGCCCTAGACCCAGGGCCCTTTGACCAGGGC600                         AGCCCCTTTGCCCAGGAGCTGCTGGACGACGGTCAGCAAGCCAGCCCCTA650                         CCACCCCGGCAGCTGTGGCGCAGGAGCCCCCTCCCCTGGCAGCTCTGACG700                         TCTCCACCGCAGGGACTGGTGCTTCTCGGAGCTCCCACTCCTCAGACTCC750                         GGTGGAAGTGACGTGGACCTGGATCCCACTGATGGCAAGCTCTTCCCCAG800                         CGATGGTTTTCGTGACTGCAAGAAGGGGGATCCCAAGCACGGGAAGCGGA850                         AACGAGGCCGGCCCCGAAAGCTGAGCAAAGAGTACTGGGACTGTCTCGAG900                         GGCAAGAAGAGCAAGCACGCGCCCAGAGGCACCCACCTGTGGGAGTTCAT950                         CCGGGACATCCTCATCCACCCGGAGCTCAACGAGGGCCTCATGAAGTGGG1000                        AGAATCGGCATGAAGGCGTCTTCAAGTTCCTGCGCTCCGAGGCTGTGGCC1050                        CAACTATGGGGGCAAAAGAAAAAGAACAGCAACATGACCTACGAGAAGCT1100                        GAGCCGGGCCATGAGGTACTACTACAAACGGGAGATCCTGGAACGGGTGG1150                        ATGGCCGGCGACTCGTCTACAAGTTTGGCAAAAACTCAAGCGGCTGGAAG1200                        GAGGAAGAGGTTCTCCAGAGTCGGAACTGAGGGTTGGAACTATACCCGGG1250                        ACCAAACTCACGGACCACTCGAGGCCTGCAAACCTTCCTGGGAGGACAGG1300                        CAGGCCAGATGGCCCCTCCACTGGGGAATGCTCCCAGCTGTGCTGTGGAG1350                        AGAAGCTGATGTTTTGGTGTATTGTCAGCCATCGTCCTGGGACTCGGAGA1400                        CTATGGCCTCGCCTCCCCACCCTCCTCTTGGAATTACAAGCCCTGGGGTT1450                        TGAAGCTGACTTTATAGCTGCAAGTGTATCTCCTTTTATCTGGTGCCTCC1500                        TCAAACCCAGTCTCAGACACTAAATGCAGACAACACCTTCCTCCTGCAGA1550                        CACCTGGACTGAGCCAAGGAGGCCTGGGGAGGGCCTAGGGGAGCACCGTG1600                        ATGGAGAGGACAGAGCAGGGGCTCCAGCACCTTCTTTCTGGACTGGCGTT1650                        CACCTCCCTGCTCAGTGCTTGGGCTCCACGGGCAGGGGTCAGAGCACTCC1700                        CTAATTTATGTGCTATATAAATATGTCAGATGTACATAGAGATCTATTTT1750                        TTCTAAAACATTCCCCTCCCCACTCCTCTCCCACAGAGTGCTGGACTGTT1800                        CCAGGCCCTCCAGTGGGCTGATGCTGGGACCCTTAGGATGGGGCTCCCAG1850                        CTCCTTTCTCCTGTGAATGGAGGCAGAGACCTCCAATAAAGTGCCTTCTG1900                        GGCTTTTTCTAAAAAAAAAA1920                                                      (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 371                                                               (B) TYPE: Amino Acid                                                          (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:                                      MetAlaAlaThrCysGluIleSerAsnIlePheSerAsnTyrPhe                                 151015                                                                        SerAlaMetTyrSerSerGluAspSerTyrLeuAlaSerValPro                                 202530                                                                        ProAlaAlaThrPheGluAlaAspAspLeuValLeuThrLeuSer                                 354045                                                                        AsnProGlnMetSerLeuGluGlyThrGluLysAlaSerTrpLeu                                 505560                                                                        GlyGluGlnProGlnPheTrpSerLysThrGlnValLeuAspTrp                                 657075                                                                        IleSerTyrGlnValGluLysAsnLysTyrAspAlaSerAlaIle                                 808590                                                                        AspPheSerArgCysAspMetAspGlyAlaThrLeuCysAsnCys                                 95100105                                                                      AlaLeuGluGluLeuArgLeuValPheGlyProLeuGlyAspGln                                 110115120                                                                     LeuHisAlaGlnLeuArgAspLeuThrSerSerSerSerAspGlu                                 125130135                                                                     LeuSerTrpIleIleGluLeuLeuGluLysAspGlyMetAlaPhe                                 140145150                                                                     GlnGluAlaLeuAspProGlyProPheAspGlnGlySerProPhe                                 155160165                                                                     AlaGlnGluLeuLeuAspAspGlyGlnGlnAlaSerProTyrHis                                 170175180                                                                     ProGlySerCysGlyAlaGlyAlaProSerProGlySerSerAsp                                 185190195                                                                     ValSerThrAlaGlyThrGlyAlaSerArgSerSerHisSerSer                                 200205210                                                                     AspSerGlyGlySerAspValAspLeuAspProThrAspGlyLys                                 215220225                                                                     LeuPheProSerAspGlyPheArgAspCysLysLysGlyAspPro                                 230235240                                                                     LysHisGlyLysArgLysArgGlyArgProArgLysLeuSerLys                                 245250255                                                                     GluTyrTrpAspCysLeuGluGlyLysLysSerLysHisAlaPro                                 260265270                                                                     ArgGlyThrHisLeuTrpGluPheIleArgAspIleLeuIleHis                                 275280285                                                                     ProGluLeuAsnGluGlyLeuMetLysTrpGluAsnArgHisGlu                                 290295300                                                                     GlyValPheLysPheLeuArgSerGluAlaValAlaGlnLeuTrp                                 305310315                                                                     GlyGlnLysLysLysAsnSerAsnMetThrTyrGluLysLeuSer                                 320325330                                                                     ArgAlaMetArgTyrTyrTyrLysArgGluIleLeuGluArgVal                                 335340345                                                                     AspGlyArgArgLeuValTyrLysPheGlyLysAsnSerSerGly                                 350355360                                                                     TrpLysGluGluGluValLeuGlnSerArgAsn                                             365370                                                                        (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:                                      CGGGATCCGCTGCAACCTGTGAGATTAGC29                                               (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 29                                                                (B) TYPE: Nucleic Acid                                                        (C) STRANDEDNESS: Single                                                      (D) TOPOLOGY: Linear                                                          (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:                                      GCAGATCTCAGTTCCGACTCTGGAGAACC29                                               __________________________________________________________________________

What is claimed is:
 1. An isolated polynucleotide comprising a memberselected from the group consisting of:(a) a polynucleotide havinggreater than 95% identity to a polynucleotide encoding a polypeptidecomprising amino acid of SEQ ID NO: 2; (b) a polynucleotide havinggreater than 95% identity to a polynucleotide encoding a firstpolypeptide which has 90% identity to second polypeptide of SEQ ID NO: 2wherein first polypeptide is capable of binding to DNA; (c) apolynucleotide which by virtue of redundancy of the genetic code encodesthe same amino acids of SEQ ID NO: 2; and (d) a polynucleotide which iscomplementary to the polynucleotides of (a), (b) or (c).
 2. Thepolynucleotide of claim 1 wherein the polynucleotide is DNA.
 3. Thepolynucleotide of claim 1 wherein the polynucleotide is RNA.
 4. Apolynucleotide of claim 2 comprising a polynucleotide having greaterthan 95% identity to a polynucleotide encoding a polypeptide comprisingamino acid of SEQ ID NO:
 2. 5. A polynucleotide of claim 2 comprising apolynucleotide having greater than 95% identity to a polynucleotideencoding a first polypeptide which has 90% identity to secondpolypeptide of SEQ ID NO: 2 wherein first polypeptide is capable ofbinding to DNA.
 6. A polynucleotide of claim 2 which by virtue ofredundancy of the genetic code encodes the same amino acids of SEQ IDNO:
 2. 7. The polynucleotide of claim 2 comprising nucleotides set forthin SEQ ID NO:
 1. 8. The polynucleotide of claim 2 which encodes apolypeptide comprising amino acids of SEQ ID NO:
 2. 9. An isolatedpolynucleotide comprising a member selected from the group consistingof;(a) a polynucleotide having greater than 95% identity to apolynucleotide encoding the same mature polypeptide expressed by thehuman cDNA contained in ATCC Deposit No. 98238; (b) a polynucleotidehaving greater than 95% identity to a polynucleotide encoding a firstpolypeptide which has 90% identity to the same mature polypeptideexpressed by the human cDNA contained in ATCC Deposit No. 98238 whereinfirst polypeptide is capable of binding to DNA; (c) a polynucleotidewhich by virtue of redundancy of the genetic code encodes the samemature polypeptide expressed by the human cDNA contained in ATCC DepositNo. 98238; and (d) a polynucleotide which is complementary to thepolynucleotides of (a), (b) or (c).
 10. A polynucleotide of claim 9comprising a polynucleotide having greater than 95% identity to apolynucleotide encoding the same mature polypeptide expressed by thehuman cDNA contained in ATCC Deposit No.
 98238. 11. A polynucleotide ofclaim 9 comprising a polynucleotide having greater than 95% identity toa polynucleotide encoding a first polypeptide which has 90% identity tothe same mature polypeptide expressed by the human cDNA contained inATCC Deposit No. 98238 wherein first polypeptide is capable of bindingto DNA.
 12. A polynucleotide of claim 9 which by virtue of redundancy ofthe genetic code encodes the same mature polypeptide expressed by thehuman cDNA contained in ATCC Deposit No.
 98238. 13. A vector comprisingthe DNA of claim
 2. 14. A host cell comprising the vector of claim 13.15. A process for producing a polypeptide comprising expressing from thehost cell of claim 14 a polypeptide encoded by said DNA.
 16. A processfor producing a cell which expresses a polypeptide comprisingtransforming or transfecting the cell with the vector of claim 13 suchthat the cell expresses the polypeptide encoded by the human cDNAcontained in the vector.